PROJECT SUMMARY Tip60 protein, also known as Tat-interactive protein 60 kD, has been associated with induction of apoptosis, the DNA damage response (DDR), and cell-cycle senescence -- functions that are not mutually exclusive -- in cell-lines and in cancer cells. We have shown that Tip60 is a vital protein in the early embryo, and that it undergoes a curious isoprotein shift as neonatal cardiomyocytes (CMs) enter replicative senescence, an event that was recently shown to be induced by activation of the DDR in these cells. Although the heart is enriched in Tip60 protein, its role in this organ remains unclear. A recent study using cultured neonatal CMs revealed that ischemia increases Tip60 content and consequent apoptosis, effects that were prevented in Tip60- depleted cells. This result is consistent with our previous finding in heterozygous Tip60+/- mice showing that modest reduction of Tip60 protein in the adult heart caused release of CMs from cell-cycle arrest while inhibiting apoptosis. Taken together, these findings suggest that reduction or inactivation of Tip60 should enhance cardioprotection during ischemia, by suppressing CM death and permitting CM regeneration. To test this possibility we have developed an animal model in which Tip60 can be conditionally depleted. Using these mice we are addressing Specific Aims to test the twofold hypothesis that Tip60 is recruited to the telomere during early neonatal stages to induce the DDR and replicative senescence in CMs (Aim 1), and that depletion or inactivation of Tip60 in the adult heart confers cardioprotection from ischemia by inhibiting apoptosis and permitting CM regeneration (Aim 2). Experimentally, the first Aim will determine whether Tip60 becomes associated with the CM telomere at early neonatal stages, and whether its depletion reduces telomere length and disrupts the DDR while extending the window of neonatal CM proliferation. The second Aim will determine whether knockdown or transient inactivation of Tip60 in the ischemic adult heart inhibits apoptosis while permitting CM regeneration. Fulfillment of this hypothesis will advance our understanding of how the post- mitotic differentiated state of CMs is attained, while establishing Tip60 as a cardioprotective target.